Disordered control of smooth muscle function is common in gastrointestinal diseases, which cost billions of dollars in health care spending each year. Gastroparesis is one of the most significant manifestations of gastrointestinal dysmotility, particularly in diabetes mellitus. However, therapeutic options are limited reflecting incomplete understanding of cellular dynamics within the neuromuscular compartment. Previous research has identified interstitial cells of Cajal (ICC) as the cell type most commonly affected in gastroparesis. ICC serve as the physiological pacemaker for phasic contractile activity and mediate cholinergic and nitrergic neuromuscular neurotransmission. Diabetic ICC depletion may arise from reduced insulin/IGF1 signaling and macrophage action, which interfere with expression of the receptor tyrosine kinase Kit. A critical gap in our knowledge is the lack of understanding of the fate of ICC lost from these insults. Cell death has been observed but not consistently, prompting investigators to hypothesize ICC transdifferentiation, which could be reversible. However, evidence of ICC survival following loss of Kit and other biomarkers has been elusive. Therefore, the main objective of this project is to identify ICC fates, their mechanisms and reversibility in diabetes. Our central hypothesis is that depletion of the ICC transcription factor Etv1 from reduced Kit signaling, together with inhibition of ?erasers? of repressive epigenetic marks due to disturbed mitochondrial metabolism, lead to decommissioning and eventual silencing of super-enhancers that normally drive the transcription of genes critical for ICC identity including Kit. These changes are accompanied by the upregulation of the closely related gene Pdgfra, conferring on the surviving ICC the identity and function of ?fibroblast-like cells? (FLC), which mediate purinergic neuromuscular neurotransmission. Specific Aim 1 is to provide definitive evidence of ICC- to-FLC phenotypic switch using in-vivo genetic lineage tracing and novel cell lines combined with phenotyping by flow cytometry, immunofluorescence, Western blotting and transcriptome sequencing in cell populations purified by fluorescence-activated cell sorting. Specific Aim 2 is to establish the epigenetic mechanisms underlying ICC dedifferentiation and phenotypic conversion into FLC. We will analyze histone and DNA marks, Etv1 binding and chromatin conformation in specific loci and genome-wide. We will perform mechanistic studies using RNA interference (RNAi), as well as in-vitro CRISPR-Cas9-mediated and in-vivo Cre-mediated genome editing combined with longitudinal analysis of gastric emptying. Specific Aim 3 is to determine the role of succinate in the epigenetic repression of ICC genes using RNAi, in-vivo genome editing and the above epigenomic and phenotyping methods. To facilitate translation of our findings, we will validate key mechanisms in human tissues and attempt to restore ICC in mice using epigenetic drugs approved for use in humans. The concept of manipulating interstitial cells via epigenetic reprogramming of cells with persistently altered transcriptional programs will change how we think about neuromuscular plasticity in health and disease.